Chronic alcohol abuse is the major cause of cirrhosis and liver failure in adult patients in the United States. Alcoholic liver disease in patients progresses from steatosis to steatohepatitis, fibrosis, and cirrhosis. The current concept is that chronic alcoholic intake increases intestinal permeability that leads to an elevation of endotoxin (lipopolysaccharide, LPS) levels in the portal vein. Increased endotoxin via the portal vein stimulates Kupffer cells through Toll-like receptor (TLR) 4, a receptor for LPS, which promotes hepatic inflammation resulting in alcoholic liver injury. Not only LPS, but also bacterial DNA levels in blood and ascites are elevated in patients with alcoholic-induced liver cirrhosis. Bacterial DNA is recognized by TLR9 that is widely expressed in immune cells including Kupffer cells, resident macrophages in the liver. On the other hand, we have recently shown that TLR4 directly activates hepatic stellate cells (HSCs) in hepatic fibrosis. We hypothesize that excessive alcohol intake disrupts intestinal epithelial barrier leads to translocation of intestinal microflora- derived LPS and bacterial DNA into the liver. LPS and bacterial DNA activate TLR4 and TLR9, respectively, expressed in Kupffer cells and HSCs, which in turn produce inflammatory and fibrogenic mediators, resulting in alcoholic steatosis, steatohepatitis (ASH) and fibrosis. Synergistic interaction between TLR4 and TLR9, and increased sensitivity of hepatocytes to cell death might further exacerbate the degrees of ASH and fibrosis. Upon ethanol treatment, Kupffer cells in the liver produce inflammatory cytokines, which could be associated with systemic organ injury including brain injury. Based on these hypotheses, the aims of this proposal are: Aim #1: To determine the role of TLR4 on the activation of Kupffer cells and HSCs in ASH; TLR4-bone marrow chimera will be generated and treated with intragastric ethanol feeding. Brain injury and intestinal permeability will be assessed. Aim #2: To determine the role of TLR9 in ASH; The responsible cell types for TLR9 in the liver will be assessed in ASH models. Aim #3: To determine the synergistic actions by TLR4 and TLR9 in Kupffer cells and HSCs. Aim #4: To determine whether the sensitivity of hepatocytes to cell death is increased in ASH. We will test these specific aims using the continuous intragastric ethanol feeding model. The proposed study will provide insight into the molecular mechanism underlying the role of multiple TLR signaling in Gut- Liver-Brain interaction in alcohol-induced pathogenesis. PUBLIC HEALTH RELEVANCE: Alcoholic steatohepatitis represents a massive health care burden worldwide. The goal of this study is to investigate whether TLR4 and TLR9 synergistically promote alcoholic steatohepatitis and subsequent brain injury. The results from this study will provide the potential targeting for the therapy of alcohol-induced organ pathogenesis including steatohepatitis and brain injury.